Hepatitis C virus (HCV) infects more than 170 million people worldwide, causing acute and chronic hepatitis and hepatocellular carcinoma. With no vaccine available to protect against HCV infection and only a subset of chronically infected patients responding to current treatment options, there is an immediate need for new effective HCV antivirals. Since its discovery in 1989, a major obstacle impeding HCV research has been the lack of robust cell culture and small animal infection models. While significant in vitro advancement has been made in the last 5 years with the development of the first robust in vitro HCV infection system, the development of a robust and widely available small animal model for pathology and immunology studies is still needed. HCV entry not only represents a promising multi-faceted opportunity for drug discovery, but we have yet to identify all the factors sufficient for HCV infection including the one(s) that dictate the restricted liver and/or species tropism of infectious HCV entry. While the recent discovery of human OCLN as a factor necessary for HCVpp entry into mouse cells appears to have brought us one step closer to understanding HCV species tropism, to date authentic HCVcc infection of non-human cells has not been achieved suggesting that other entry factors, specific for HCVcc, still exist. Our long term goal is to understand the cellular and viral factors that mediate HCVcc entry in order to identify novel molecular targets for therapeutic intervention and facilitate the much needed development of small animal models of HCV infection. As such, the objective of this R21 application is to elucidate the role of a newly identified putative HCV entry factor. Specifically, we hypothesize that the cellular transferrin receptor 1 (TfR1) is an HCV entry receptor and that it may be responsible for the restricted tropism of HCV entry. This hypothesis is based on published precedents demonstrating that TfR1 functions as a species specific receptor for other viruses and our preliminary data which demonstrate that TfR1 is required for HCV entry. Hence, we will pursue the following two specific aims: 1) To characterize how and when TfR1 participates in HCV entry by determining when TfR1 acts in the HCV entry process and if that interaction involves direct TfR1 binding to HCV and 2) To determine whether TfR1 is a determinant of HCV tropism by characterizing a panel of cells to determine if TfR1 expression confers HCVcc permissiveness to non-hepatic or non-human cells. Taken together, this information will lay the groundwork that will enable a more detailed and specifically focused R01 application aimed at thoroughly understanding HCV TfR1-dependent entry and/or the development of an infectious HCV mouse model.